Affiliations
Department of Entomology and Nematology, University of Florida,
Gainesville, Florida, United States of America,
USDA/ARS, Center for Medical, Agricultural and Veterinary Entomology,
Gainesville, Florida, United States of America

Affiliations
Department of Microbiology and Molecular Genetics, School of Medicine,
University of California, Irvine, California, United States of
America,
Department of Molecular Biology and Biochemistry, University of
California, Irvine, California, United States of America

Figures

Abstract

Transposon-mediated transformation was used to produce Anopheles
stephensi that express single-chain antibodies (scFvs) designed to
target the human malaria parasite, Plasmodium falciparum. The
scFvs, m1C3, m4B7, and m2A10, are derived from mouse monoclonal antibodies that
inhibit either ookinete invasion of the midgut or sporozoite invasion of
salivary glands. The scFvs that target the parasite surface, m4B7 and m2A10,
were fused to an Anopheles gambiae antimicrobial peptide,
Cecropin A. Previously-characterized Anopheles cis-acting DNA
regulatory elements were included in the transgenes to coordinate scFv
production with parasite development. Gene amplification and immunoblot analyses
showed promoter-specific increases in transgene expression in blood-fed females.
Transgenic mosquito lines expressing each of the scFv genes had significantly
lower infection levels than controls when challenged with P.
falciparum.

Author Summary

Malaria eradication will require vector-control strategies that are both
self-sustaining and not affected by migration of infected humans and mosquitoes.
Replacement of wild malaria-susceptible mosquito populations with transgenic
strains refractory to parasite development could interrupt the cycle of disease
transmission and support eradication efforts. Production of P.
falciparum-resistant mosquitoes is a necessary first step towards
investigating the population replacement strategy. Here we show that An.
stephensi engineered to produce P.
falciparum-targeting effector molecules are resistant to this important
human malaria parasite. Two of the three effector molecules represent a novel
combination of components derived from the immune systems of mosquitoes and
mice. An important feature of these molecules is that they are unlikely to
significantly harm the mosquito, as the mosquito component is an
Anopheles antimicrobial peptide with activity against
Plasmodium, while the other component is based on a murine
antibody selected for its ability to bind specifically to a parasite protein.
Transgenes with this design coupled with a gene-drive system could be used
alongside vaccines and drugs to provide sustainable local elimination of malaria
as part of a long-term strategy for eradication.

Funding: The research was supported by grants from the NIH NIAID (AI29746) to AAJ,
(R01AI45999) and (K24AI068903) to JMV, the Natural Science Foundation of China
(No. 30771871) to XC, and an Academic Senate research development grant from
UCSD to FL. The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.

Competing interests: The authors have declared that no competing interests exist.

Introduction

Plasmodium falciparum, a causative agent of human malaria, is a
vector-borne parasite that is responsible for more than 500 million clinical disease
cases each year [1].
The selection of insecticide-resistant mosquitoes and drug-resistant parasites
necessitates an ongoing search for new disease-control methods. A proposed strategy
for interrupting transmission is to replace wild, malaria-susceptible mosquito
populations with transgenic, Plasmodium-resistant mosquitoes [2]–[4]. Key
components of this approach are effector molecules that inhibit parasite development
when expressed from a transgene. The mechanisms by which effector molecules function
can vary greatly, as the development of the malaria parasites within mosquitoes
involves several transitions of environment, physiology and morphology [5].

When mosquitoes feed on infected humans, they ingest parasites in the form of
gametocytes. These produce gametes that fuse to form diploid zygotes that develop
into the motile ookinetes. The ookinetes invade and traverse the mosquito midgut
epithelium and then rest beneath the basal lamina of the midgut, forming oocysts.
Thousands of sporozoites develop within the oocysts before budding out into the
circulatory fluid, the hemolymph, and invading the salivary glands. Several effector
molecules have been tested for their ability to target the parasite during either
early sporogony in the midgut, or late sporogony in the hemolymph or salivary glands
[5]–[8]. An effector mechanism based on the mosquito signaling
protein Akt is the only one to date shown to inhibit completely P.
falciparum development in a transgenic Anopheles
mosquito [7].

One effector molecule strategy exploits the finding that monoclonal antibodies (mAbs)
that recognize surface-bound or secreted parasite molecules can inhibit pathogen
development [9]–[14]. Two mAbs, 4B7 and 1C3, target parasites early in their
development within mosquitoes. 4B7 binds P. falciparum surface
protein Pfs25, a molecule expressed on the surface of ookinetes, and inhibits
parasite development completely when fed to Anopheles mosquitoes in
a gametocytemic bloodmeal [9]. In contrast, 1C3 binds a parasite-secreted enzyme,
P. falciparum chitinase 1, and inhibits oocyst formation of
P. falciparum when incorporated into infectious bloodmeals
[10]. A third mAb,
2A10, binds P. falciparum circumsporozoite protein (CSP), and when
pre-incubated with sporozoites, greatly decreases their ability to infect cultured
hepatocytes [11], [12].

Although the size and complexity of mAbs exclude them from consideration as potential
effector molecules, single-chain antibodies (scFvs), which retain the binding
specificity of a mAb, are much smaller and can be produced from a single
transcription unit [15]. An scFv targeting the P. gallinaceum
CSP inhibited sporozoite invasion of salivary glands in Aedes
aegypti in both transient assays and transgenic mosquitoes [13], [16].
Anopheles stephensi fed Escherichia coli
expressing an anti-P. berghei scFv-immunotoxin were shown to have
significantly-reduced oocyst densities when fed on parasite-infected mice [14]. Furthermore,
an scFv derived from the 1C3 mAb reduced significantly P.
falciparum parasite transmission to mosquitoes [17]. The experiments described in the
work presented here test the scFv-based strategy on human malaria parasites in
transgenic mosquitoes and support the further development and evaluation of these
molecules as disease-control tools.

scFvs based on the 1C3, 4B7 and 2A10 mAbs were expressed in transgenic An.
stephensi and their efficacy tested in parasite challenge assays with
P. falciparum. Anopheles stephensi was chosen
because it is a significant vector of urban malaria transmission in the Indian
subcontinent and is an efficient model for transgenic research. To distinguish the
novel scFvs developed in this study, we refer to them as “modified” 1C3,
4B7 or 2A10 (m1C3, m4B7, m2A10). For the m4B7 and m2A10 transgenes, the An.
gambiae Cecropin A gene (AgCecA) was joined to the
scFv gene to form a single open reading frame (ORF). Cecropin A is an antimicrobial
peptide that has microbiocidal activity against both gram-negative and gram-positive
bacteria, as well as multiple Plasmodium species [18], [19]. This broad
activity is due to its ability to form large pores in cell membranes [20]. With the
addition of cecropin A, the m4B7 and m2A10 scFvs possess both parasite-binding and
antimicrobial activity. The cecropin A peptide was not joined to m1C3 as the target
of this scFv is a secreted molecule [17].

Anopheles gambiae carboxypeptidase A (AgCPA[21], [22]) gene regulatory
sequences were included in m4B7 and m1C3 transgenes to coordinate their expression
with the development of ookinetes. Anopheles stephensi vitellogenin
1 (AsVg1[23]) regulatory
elements were joined to the m2A10 scFv to direct transgene expression in the female
fat body. Thus, m2A10 secreted from the fat body into the hemolymph could encounter
sporozoites migrating to the salivary gland. When challenged in multiple experiments
with P. falciparum infectious gametocyte cultures, scFv-expressing
transgenic lines displayed statistically-significant, reduced mean intensities of
infection and in most trials lower parasite prevalence when compared to control
mosquitoes.

Results

Transgene assembly, transgenesis, and gene copy-number analyses

The scFv genes were synthesized commercially to incorporate either the
AgCPA signal sequence or the entire AgCecA
ORF (Figure 1). Codons
corresponding to the amino acids serine, proline, alanine, threonine, and
arginine displayed the greatest frequency bias differences between Mus
musculus and An. gambiae (Table S1)
[24],
and these were replaced in the mouse-derived scFv sequences by those favored by
the mosquito. DNA sequence encoding a short polypeptide linker (five amino
acids) was used to join the heavy- and light-chain variable fragments of m4B7
and m2A10 scFvs and a longer linker (encoding 15 amino acids) joined the two
corresponding moieties of m1C3. Long linkers permit intramolecular pairing of
variable fragments, while short linkers favor the intermolecular joining of scFv
molecules to form multimers containing multiple antigen recognition sites [25]. The m1C3
and m4B7 scFv genes were joined to AgCPA regulatory elements
and inserted into a pBac [3xP3-EGFP] plasmid to construct the
transformation vectors (Figure
2). Similarly, the m2A10 scFv gene was joined to
AsVg1 regulatory elements and inserted into a pBac
[3xP3-dsRed] plasmid.

A mature mouse immunoglobulin molecule consists of two heavy- and
light-chain polypeptides each linked through disulfide (ss) bonds (top
image). The single-chain antibodies are composed of the variable regions
(Fv) of the heavy (VH) and light (VL) chains (gray
and open boxes, respectively) of a mouse monoclonal antibody. The m4B7
and m2A10 scFv transgenes encode a short polypeptide linker of 5 amino
acids (5aa) between VH and VL. These transgenes
include sequence for a long polypeptide linker of 15 amino acids (15aa)
joining the VH to the An. gambiae Cecropin A
peptide (CecA), including its signal sequence. The VH region
present in the m1C3 transgene is joined to the An. gambiae
Carboxypeptidase A gene signal sequence, and joined by a
long polypeptide linker to the VL region. Select codons in
the variable region genes were codon-optimized to facilitate efficient
translation. Anopheles promoter sequences (P) were
joined to the scFvs to direct tissue-specific transgene expression.

(A) Schematic representations of the single-chain antibody (scFv)
transformation constructs. The scFv heavy (VH) and light
(VL) variable region genes in the m1C3 construct are
joined by sequence encoding a long polypeptide linker (multiple grey
boxes). The sequence encoding the VH is joined to the
An. gambiae carboxypeptidase A signal sequence
(AgCp sig). In the m4B7 and m2A10 constructs, the
An. gambiae Cecropin A gene
(CecA), including signal sequences
(CecA sig), is joined by sequence encoding a long
polypeptide linker to the scFv VH and VL genes.
The VH and VL genes are joined by a short
polypeptide linker (single grey box). All three scFvs are joined to an
epitope tag (E tag). The CecA-m2A10 effector gene is flanked by
An. stephensi vitellogenin 1 regulatory sequences
(AsVg 5′ UTR, 3′UTR), while the m1C3
and CecA-m4B7 genes are flanked by AgCP A regulatory
sequences (AgCp 5′ UTR, 3′UTR). A
transformation marker (EGFP or DsRed) joined to the Pax3 (3xP3) promoter
and the SV40 polyadenylation sequence also is contained between the
piggyBac transposase arms (pBacLH, RH). Select
restriction endonuclease sites present in the transgene are indicated in
the diagram. Probes used to identify the integrated transgenes are
indicated by horizontal bars above each schematic representation. (B)
Genomic DNA from m1C3 and wild-type control females was digested and
hybridized to either an EGFP probe (left) or an m1C3 probe (right). (C)
Genomic DNA from m4B7 and wild-type control females was digested and
hybridized to either an EGFP probe (left) or an
m4B7/AgCPA 3′UTR probe (right). (D) Genomic
DNA from m2A10 and wild-type control females was digested and hybridized
to an m2A10 probe. The restriction endonucleases used in each experiment
are listed above the blot, and the identity of each transgenic line is
listed above each lane. The locations of molecular weight markers are
indicated in kilobase pairs (kb).

The three transformation plasmids pBac [3xP3-EGFP]-m1C3, pBac
[3xP3-EGFP]-m4B7 and pBac [3xP3-dsRed]-m2A10 were injected
into 980, 615 and 765 embryos, respectively. Three transgenic m1C3 mosquito
lines (21.1, 39.1 and P4.1) were established from EGFP-positive families derived
from 78 surviving adults. Two transgenic m4B7 mosquito lines (25.1 and P6.1)
were established from 89 adults, and seven transgenic m2A10 mosquito lines
(18.1, 20, 34.1, 39.1, 44.1, P5.1 and P7.1) were established from 105
adults.

Southern blot analyses were used to verify transgene insertions and to determine
the number of integrated constructs in each line (Figure 2). Hybridization of an m1C3 probe to
genomic DNA digested with both SphI and XhoI
restriction endonucleases produced a diagnostic fragment of ∼1.2 kilobase
pairs (kb) in transgenic samples, confirming m1C3 integration. Genomic DNA
digested with SpeI and hybridized to an EGFP probe produced
multiple fragments in each transgenic sample, indicating that there were at
least three, nine, and ten copies in lines 21.1, 39.1, and P4.1, respectively.
Genomic DNA digested with ApaI, AscI, and
FseI, and hybridized to a probe complementary to the m4B7
gene and the AgCPA 3′UTR produced two diagnostic
fragments of 940 and 810 base pairs (bp), verifying transgene insertion. A
second blot, comprising XhoI-digested genomic DNA recovered
from transgenic mosquitoes and hybridized with a 3XP3 EGFP probe, revealed
several fragments in each sample, indicating that at least four copies of the
m4B7 transgene were present in each line. Lastly, genomic DNA digested with both
XbaI and BamHI and hybridized to an m2A10
probe produced an ∼1 kb diagnostic fragment in each transgenic sample. The
same probe hybridized to HindIII-digested genomic DNA bound
multiple DNA fragments in each m2A10 sample, indicating the presence of six,
three, three, four, six, seven and three copies in transgenic lines 18.1, 20,
34.1, 39.1, 44.1, P5.1, P7.1, respectively. Transgenic lines were maintained by
intercrossing at each generation. However, selection pressures on individual
transgene insertions, small founding colony sizes and independent assortment
likely result in loss over time of some of the insertions.

Characterization of transgene expression

Reverse-transcriptase-PCR (RT-PCR) and Real-time quantitative RT-PCR (RT-qPCR)
were used to evaluate the presence and relative abundance of m1C3, m4B7 or m2A10
transcription products in non-blood-fed and blood-fed mosquitoes in all of the
established transgenic lines. No significant correlation was seen between
transgene copy number and amount of transcription product detected (data not
shown). Therefore, the lines m1C3 P4.1, m4B7 25.1 and m2A10 44.1, each of which
displayed the highest levels of transcript accumulation in their respective
group, were selected for use in all further analyses. Southern blot analyses of
the generations of m1C3 P4.1, m4B7 25.1 and m2A10 44.1 used in the challenge
assays indicated the presence of eight, four, and four copies of the respective
transgenes.

Transgene-specific transcript accumulation profiles detected by RT-PCR were
similar in mRNA samples prepared from the dissected midguts of m1C3 P4.1 and
m4B7 25.1 females (Figure
3). Both lines showed constitutive accumulation in midguts from non-blood
fed mosquitoes. In addition, each line showed accumulation of their respective
mRNAs at 4 hours post-bloodmeal (hPBM), and signals were evident at 12 and 24
hPBM. m4B7 transcript also could be detected at low levels at 48 hPBM. No
amplification products were produced from mRNA prepared from female carcasses or
males of each line. As expected, control reactions using mRNA from midguts
dissected at 4 hPBM from wild-type, non-transgenic females were negative.
RT-qPCR analysis at multiple post-bloodmeal time points was used as an
independent measure of m1C3 P4.1 expression. The highest measured level of m1C3
mRNA, 10,000-fold above the control level, was observed in the 16 hPBM midgut
sample (paired T-test, one tailed p-value = 0.005), but
similar elevated levels also were seen at 24 and 48 hPBM. At this time, we
cannot account for the difference in the RT-PCR and RT-qPCR results at 48 hPBM,
although this could result from individual females that responded differentially
to the feeding regimen. This difference is not expected to have affected the
outcome of the challenge experiments because this scFv targets parasites within
the first 24 hPBM.

Representative results of gene amplification experiments show tissue- (A,
B, C), sex- (A, B, C) and stage-specific (C) accumulation of transgene
transcription products. (A) RT-PCR was used to detect m4B7 and
endogenous An. stephensi carboxypeptidase A
(AsCPA) transcript in RNA isolated from either
whole m4B7 25.1 transgenic males (M) or dissected wild-type (WT)
control, non-blood-fed m4B7 25.1 transgenic females (NBF) and m4B7 25.1
transgenic females at multiple post-bloodmeal time points (indicated
above each lane). For female samples, RNA was extracted from either gut
or carcass homogenates. (B) RT-PCR was used to detect m1C3 and
endogenous An. stephensi carboxypeptidase A
(AsCPA) transcript as described in (A). The
An. stephensi S26 ribosomal protein transcript
(rpS26) was amplified as a loading control in all
RT-PCR experiments. (C) Accumulation of m2A10 44.1 and endogenous
An. stephensi vitellogenin (AsVg1)
transcript in transgenic males, non-blood-fed females, and females at
multiple post-bloodmeal time points was assayed by RT-PCR. RNA was
isolated from adult whole-body homogenates of wild-type control and
m2A10 44.1 transgenic mosquitoes. RNA from wild-type control females was
included as both a positive control for AsVg1
expression and as a negative control for m2A10 44.1 amplification.
Parallel reactions in which the reverse transcription reaction step were
omitted demonstrated that the observed amplification products originated
exclusively from RNA (data not shown). (D) Developmental expression of
m1C3 mRNA. m1C3 mRNA abundance in the midguts of females is described as
transcript numbers in log-scale. Each data point represents the average
of three independent biological replicates. An asterisk indicates a
significantly larger number of transcripts when compared to NBF (paired
T-test, one tailed p-value, 16 h p = 0.0050, 24 h
p = 0.0067, 48 h
p = 0.0018).

Immunoblot analyses of m1C3 and m4B7 transgenic mosquitoes were unproductive
despite repeated attempts. Although high levels of proteinase inhibitors were
used during sample preparation, it is possible that the transgene products were
degraded quickly in the strong digestive milieu of the post-feeding midgut
lumen.

Transgene transcripts detected in whole transgenic m2A10 44.1 females showed sex-
and stage-specificity (Figure
3). No signals were seen in samples derived from mRNA prepared from
males and non-blood fed transgenic or control wild-type, non-transgenic females.
Specific transcript accumulation was evident in m2A10 44.1 females at 12 and 24
hPBM in an expression pattern similar to that of endogenous
AsVg1, but was not as abundant at 48 hPBM.

While expression of the m1C3 and m4B7 scFvs is necessary only during the first 24
hours post-bloodmeal, expression of m2A10 must be sustained over several days,
as oocysts can mature asynchronously [26]. Denaturing immunoblot
analyses were performed on m2A10 44.1 females sampled over the course of four
bloodmeals to evaluate whether protein expression could be induced repeatedly
(Figure 4; Figure S1).
Anti-E tag antibody specifically detected a polypeptide with an approximate
Mr of 32 kiloDaltons (kDa), consistent with the predicted size of
m2A10 protein, in transgenic blood-fed females at 24, 48, 72 and 96 hPBM. The
continuous presence of m2A10 was detected in females that were given bloodmeals
once every five days. Expression of m2A10 also was observed at 12 hPBM in
additional immunoblot analyses (data not shown). Immunoblots of hemolymph
samples indicated that m2A10 protein was present in the hemolymph of blood-fed
transgenic females (Figure
4). Immunoblot analyses of hemolymph samples analyzed in non-denaturing
conditions detected m2A10 protein in several multimeric conformations with
estimated Mrs of 125, 223, 284, and 485 kDa.

(A) Immunoblot analyses of transgenic m2A10 44.1 An.
stephensi. An anti-E tag antibody was used to detect m2A10
protein in whole body homogenates of transgenic females. Purified m2A10
scFv protein, which contained an E tag but did not contain Cecropin A,
was used as a positive control (+). Female mosquitoes were given
bloodmeals (bm) once every five days, and examined at 24, 48, 72, and 96
hours after each meal. (B) An immunoblot of both whole females (W) and a
hemolymph preparation of females (H) from transgenic m2A10 44.1 and
wild-type control mosquitoes (WT) 48 hours post-bloodmeal. Anti-E tag
antibody was used for detection of m2A10. (C) An immunoblot of m2A10 and
wild-type control female hemolymph samples, prepared in non-denaturing
conditions (left panel) and probed with anti-E tag antibody.
Non-bloodfed (NBF) and 24 hours post-bloodmeal (24hPBM) females were
compared. NativeMark protein standard (Invitrogen) was used for
molecular weight estimation. Control images of the Coomassie-stained
gels are provided in Figure S1.

Plasmodium falciparum challenge of transgenic and control
mosquitoes

Parasite challenge experiments were performed to test the efficacy of the
anti-pathogen effector molecules. Transgenic and control mosquitoes ingested
blood containing P. falciparum gametocytes through a
membrane-feeding apparatus. Control mosquitoes for most experiments were
non-transgenic (wild-type) mosquitoes. In addition, the oocyst prevalence and
mean intensities of infection of a group of m2A10 44.1 females were examined for
each challenge experiment to determine whether transgenesis alone had an impact
on parasite development.

The effect of transgene expression on parasite development for both the m4B7 25.1
and m1C3 P4.1 transgenic lines was measured by comparing the number of oocysts
in transgenic and control mosquito midguts at nine days after the infectious
bloodmeal (Table 1; Figure 5). The mean
intensities of oocyst infection were reduced by 37–81% in three
challenge experiments (1, 2 and 3, Table 1) of m4B7 25.1. However, the mean
intensities of infection were reduced by only 29–36% in two
experiments (4 and 5, Table
1) in which control mosquitoes had greater than 17 oocysts per
midgut. Mean intensities of oocyst infection were reduced by 47–73%
in mosquitoes expressing m1C3 when compared to controls. Furthermore, with the
exception of the high infection-level experiments (4 and 5, Table 1), both m1C3 P4.1 and
m4B7 25.1 transgenic mosquitoes had lower prevalence of infections than
controls.

Box plots display the observed distribution of oocyst per midgut or
sporozoite per salivary gland pair values for wild-type control
mosquitoes and m1C3 P4.1, m4B7 25.1, or m2A10 44.1 transgenic
mosquitoes. The boxes in each panel represent from bottom to top the
25th to 75th percentiles, while the vertical
lines and small horizontal bars delimit the minimum and maximum values.
Horizontal bars within the boxes indicate the median value of each
group. (A) Plots of data from all challenge experiments with m4B7 25.1
and m1C3 P4.1 mosquitoes. (B) The plot represents data from m2A10 44.1
experiments 1–4 and 5–7 separately, as indicated.

Parasite challenge assays of m2A10 44.1 involved dissecting 7–11 mosquitoes
of each group 10 days after the infectious bloodmeal to count midgut oocysts and
to confirm that both transgenic and control mosquitoes were infected
successfully (Table 2). No
statistically significant difference in the number of oocysts between transgenic
m2A10 44.1 and control mosquitoes was observed (Mann-Whitney U
test, one-tailed P value, 0.24<p<0.48). The remaining mosquitoes
(n = 8–50) in each group were examined 17–19
days after infection for the presence of sporozoites in the salivary glands
(Table 2; Figure 5). All mosquitoes were
provided an uninfected bloodmeal every five days to maintain expression of
m2A10. Engorged and un-engorged females were not separated after the uninfected
bloodmeals in experiments 1, 2, 3 and 4, and a 52–84% reduction in
mean intensity of sporozoite infection was observed in transgenic mosquitoes
when compared to the controls. To obtain a more precise measurement of the
effect of m2A10 expression upon P. falciparum development, an
additional three experiments (5, 6 and 7) were performed in which un-engorged
females were discarded after each uninfected bloodmeal. A 96–97%
reduction of mean intensity of infection was observed in m2A10 44.1 mosquitoes
that fed every five days. Furthermore, m2A10 44.1 mosquitoes in experiment 7 had
a 14% prevalence of infection compared to 78% observed in the
corresponding control.

Discussion

Previous evaluations of mosquitoes engineered genetically to express
anti-Plasmodium effector genes featured analyses of transgene
copy numbers, transgene transcription levels, detection of transgene effector
proteins, binding of effector molecules to the target parasite stage and a phenotype
of reduced parasite mean intensities of infection and prevalence [7], [8], [16], [22], [27]–[29]. Remarkably, no
single study includes all of these data and the emphasis has been on the impact of
transgene presence on parasite numbers. Expression of the two midgut-directed scFvs,
m1C3 and m4B7, was detected by RT-PCR, but not by immunoblots. The rapid degradation
of these scFvs in the midgut environment may have inhibited immunoblot detection.
However, the observation that m4B7 25.1 and m1C3 P4.1 transgenic mosquitoes have
reduced parasite loads supports the conclusion that these scFvs are expressed in the
midgut. Both transgene transcription and translation products were detected in m2A10
44.1 mosquitoes. The finding that the immunoblot analyses of non-denatured m2A10
44.1 samples detected the presence of scFv multimers is consistent with the
expectation that the short polypeptide linker joining the VH and
VL regions promotes intermolecular scFv interactions. The size of
these multimers was similar to the predicted sizes of m2A10 multimers comprising
four, seven, nine, and fifteen scFv molecules. Such scFv multimers are reported to
have high affinity to target epitopes [25].

Both m1C3 and m4B7 expressed in transgenic lines P4.1 and 25.1, respectively,
inhibited parasite development during early sporogony, resulting in significantly
reduced mean intensities of oocyst infection in eight of ten challenge experiments.
The results of two of the m4B7 25.1 challenge experiments are consistent with the
interpretation that there is a threshold level of initial parasite density above
which this scFv, at the levels expressed in these transgenic lines, cannot
efficiently inhibit ookinete development. The finding that m2A10 44.1 and wild-type
control mosquitoes did not differ in midgut infection supports the conclusion that
transgene integration alone does not necessarily impair parasite development.

When expression of m2A10 in line 44.1 was induced repeatedly by blood feeding, a
highly significant decrease in sporozoite load was observed in transgenic mosquito
salivary glands. For this transgenic line, the greatest reduction in prevalence was
found in an experiment in which the mean intensity of oocyst infection was low. It
is likely that these scFvs would effectively impair P. falciparum
transmission in field conditions, as infected wild-caught An.
gambiae carry few oocysts. Studies of An. gambiae by
Billingsley et al.[30] and
Taylor [31] found
mean numbers of oocysts per infected mosquito of 1.55 and 3.38, respectively.

Incorporation of multiple transgenes is typical for
piggyBac-mediated insertions into An. stephensi[22], [23], [32]. Although it is
reasonable to expect that higher transgene copy numbers should yield higher
expression levels, no statistically-significant correlations have been reported. We
hypothesize that many of the multiple copies have little or no expression as a
result of position effects, and that the majority of transgene expression comes from
single or small numbers of the transgenes. To mitigate copy-number issues, we have
used piggy-Bac-mediated transposition to integrate target sites for
φC31 site-specific recombination into multiple locations
in the An. stephensi genome and are now testing individual lines
for permissiveness for optimum transgene expression [33]. These lines have the added
benefit of having been evaluated for the impact on fitness of the introduced
exogenous DNA at the specific insertion site, and therefore the effects of
anti-pathogen transgene product expression can be measured directly. Furthermore, we
are eager to evaluate the phenotype of dual transgenes, for example, those combining
m4B7 and 2A10 or m1C3 and m2A10, on parasite mean intensities of infection and
prevalence. Additional studies facilitated by this approach could include testing
alternate gene regulatory sequences, such as those of the salivary gland-specific
anopheline antiplatelet protein or the An. gambiae adult
peritrophic matrix protein 1, to measure the effect of different transgene
expression patterns upon parasite development [34], [35].

Although the scFvs in lines m1C3 P4.1, m4B725.1, and m2A10 44.1 inhibited parasite
development significantly, no transgenic line displayed zero prevalence of
infection. It has been demonstrated in an avian malaria model system comprising the
vertebrate host, Gallus gallus, the mosquito host, Aedes
aegypti, and the parasite, P. gallinaceum, that
mosquitoes containing as few as 20 sporozoites in their salivary glands infected
chickens during a blood meal [16]. This finding supports the conclusion that a target of
zero prevalence is necessary for a transgenic mosquito to be incapable of disease
transmission in this system. These results are in contrast to reports of experiments
with transgenic mosquitoes and a rodent malaria parasite, P.
berghei, in which the effector molecules SM1, PLA2 and CEL-III show a
significant inhibition of parasite development (81.6%, 87% and
84.8%, respectively) [22], [27], [28]. Reductions of mean intensities of P.
berghei sporozoite infection in salivary glands below ∼400 were
sufficient to block transmission. In contrast, experimental infections of humans
with P. vivax showed that 10 sporozoites were sufficient to cause
malaria [36].
We have opted to take the conservative approach and are attempting to achieve zero
prevalence of human parasites in mosquito salivary glands [16].

Two anti-Plasmodium effector molecule strategies have yielded
transgenic mosquitoes with zero prevalence: expression of the signaling molecule
Akt, and expression of a combination of Cecropin A and Defensin A [7], [29]. The latter
study was conducted with the P. gallinaceum/Ae.
aegypti/G. gallus model system. The study of Akt
demonstrated the feasibility of producing an Anopheles mosquito
that is completely resistant to P. falciparum, however this
effector molecule may not be an optimal component of a population replacement
strategy as these mosquitoes have a significantly reduced lifespan [7]. A
synthetic peptide designed to interact with P. yoelii reduced
midgut infections of this parasite by 67–87% in An.
gambiae but was considerably less efficacious against P.
falciparum[8].
Quantitative comparisons of the efficacy of alternative effector molecules are
hindered currently by differences in expression that result from variations in
transgene location and copy number. Site-specific recombination approaches will
allow such evaluations in well-characterized ‘docking-site’ mosquito
strains [8],
[33].

The finding that m2A10 44.1 mosquitoes display up to 97% decreases in the mean
intensity of P. falciparum infection, as well as decreased
prevalence of infection, supports the argument that this scFv may be an effective
component of a malaria resistance transgene. The m1C3 and m4B7 scFv genes conferred
significant reductions in mean intensities of infection, and if expressed in higher
quantities, also may be used in the design of a transgenic, parasite-resistant
mosquito. Furthermore, expressing the scFv transgenes in additional malaria vectors,
in particular, An. gambiae, and challenging these with a variety of
P. falciparum isolates would help evaluate whether these
effector molecules could be used in multiple transmission areas.

The discovery and characterization of several effector molecules that completely
inhibit P. falciparum development will support the engineering of
mosquitoes that express multiple effector molecules. Such mosquitoes may have a
reduced likelihood of selecting for resistant parasites. Along with vaccines, drugs,
and insecticide-treated nets, parasite-resistant transgenic mosquitoes would be a
useful component in a malaria-control strategy, especially in regions where existing
interventions have been unable to eliminate disease transmission.

Materials and Methods

Mosquito rearing and maintenance

A colony of Anopheles stephensi (gift of M. Jacobs-Lorena, Johns
Hopkins University) bred in our insectary for >5 years was used in the
experiments. The mosquitoes were maintained in conditions that maximize larval
nutrition, and adult size and fitness [37]. These conditions include
maintenance of cultures at 27°C with 77% humidity and 12 hr
day/night, 30 min dusk/dawn lighting cycle. Larvae were fed a diet of powdered
fish food (Tetramin) mixed with yeast. Adults were provided water and raisins
ad libitum. Anesthetized chickens, mice, or rabbits were
used for blood feeding. Transgenic and wild-type control mosquitoes used in
parasite challenge experiments were reared in parallel using standardized
insectary procedures.

Ethics statement

This study was carried out in strict accordance with the recommendations in the
Guide for the Care and Use of Laboratory Animals of the National Institutes of
Health. The protocol was approved by the Intuitional Animal Care and Use
Committee of the University of California, Irvine (NIH Animal Welfare Assurance
number: A3416.01 (approved February 20, 2008), Protocol Number: 1998- 1411
(approved May 21, 2010). The vertebrates used as bloodmeal donors for mosquitoes
were anesthetized on a regimen that avoids the build-up of drug tolerance, and
all efforts were made to minimize suffering.

scFv sequence modifications

The sequences of the 4B7 and 2A10 variable heavy- and light-chain regions
(VH and VL, respectively) were derived from cDNA
synthesized from 2A10 and 4B7 hybridoma cell lines (obtained from E. Nardin
[New York University], and the Malaria Research and Reference Reagent
Resource Center, respectively). 2A10 cDNA was synthesized from total RNA
isolated from the hybridoma cell line using primers designed from the known
VH and VL sequence [38]. VH and
VL cDNA from the 4B7 hybridoma cell line was amplified from its
total RNA using the heavy and light primer mixes respectively, provided in the
Mouse ScFv Module/Recombinant Phage Antibody System (Amersham Biosciences). The
modified scFv genes, including either AgCPA signal sequence or
the entire AgCecA ORF, were synthesized commercially (Epoch
Biolabs) to allow for incorporation of novel features. The variable regions of
4B7, 2A10, and 1C3 [17] were optimized by replacing the codons corresponding
to the amino acids serine, proline, alanine, threonine, and arginine in the
mouse-derived sequences with those favored in An. gambiae
(Table
S1) [24]. For the m4B7 and m2A10 scFvs, the variable regions
were joined by sequence encoding a short polypeptide linker, G4S. The
VH region of the m2A10 and m4B7 scFv sequences were joined to the
AgCecA protein-coding sequence by a long polypeptide linker,
(G4S)3[39]. The
variable regions of m1C3 were joined by the same long polypeptide linker. The
m2A10 VL was joined to sequence encoding a complete E tag, while the
VL of m4B7 and m1C3 were joined to sequence encoding a partial E
tag. For m4B7 and m1C3, the remaining E tag coding sequence was joined to the
partial E tag at a later cloning step.

Transformation plasmid assembly

The pBacDsRed-AsVg5′-m2A10′-AsVg3′ plasmid was produced in two
cloning steps. First, m2A10 sequence from the commercially-synthesized pBSKm2A10
plasmid replaced the CFP gene of pSLfa-AsVg5′-CFP-AsVg3′ [23] using
XbaI and BamHI sites. Second, the
AsVg5′-m2A10-AsVg3′ sequence was joined to pBacDsRed [37] using
AscI sites. A pSLfa-AgCP5′-4B7-AgCP3′ plasmid
supplied the AgCP regulatory sequences, as well as a partial E tag sequence, for
both the pBacEGFP AgCP5′-m1C3-AgCP3′ and the pBacEGFP
AgCP5′-m4B7-AgCP3′ plasmids. The pSLfa-AgCP5′-4B7-AgCP3′
plasmid was cloned in several steps. First, the Mouse ScFv Module/Recombinant
Phage Antibody System (Amersham Biosciences) was used to produce a single chain
antibody from VH and VL cDNA from the 4B7 cell line. The
scFv was cloned into the pCANTAB 5E vector in frame with the E-Tag at the C
terminus. BclI sites were added to both ends of 4B7 scFv by
amplification using primers 4B7BclF [5′-CGTGATCAGTGAAGCTGGTGGAGTCT-3′] and
4B7BclR [5′-CGTGATCACTATGCGGCACGCGGTT-3′] from the
5′ and 3′ end and cloning into pCR4Blunt-TOPO. The
pGEMT[AgCP-SM1] plasmid, containing AgCP (AGAP009593) regulatory
regions, was generously provided by Dr. Marcelo Jacobs-Lorena [22]. The
BclI-cut 4B7 scFv fragment from TOPO
[4B7BclI] was sub-cloned into the
Bam HI sites of pGEMT[AgCP-SM1] thereby swapping
the SM1 fragment with 4B7. AgCP5′-4B7-AgCP3′ was cloned subsequently
into pSLfa1180fa [40] using enzymes SacII and
SalI. ApaI and SgrAI were
used to replace the 4B7 region of pSLFA-AgCP5′-4B7-AgCP3′ with the
commercially-synthesized m4B7 gene. AgCP5′-m4B7-AgCP3′ sequence was
then joined to pBacEGFP [40] using a 5′ blunt ligation of
AscI and KpnI sites, and a 3′
FseI ligation. To assemble the m1C3 transformation plasmid,
the enzymes ApaI and BamHI were used to
replace the 4B7 region of pSLfa-AgCP5′-4B7-AgCP3′.
AgCP5′-m1C3-AgCP3′ was then joined to pBacEGFP using
AscI restriction sites.

Microinjection and southern hybridization analyses

Microinjection of the pBac [3xP3-EGFP]-m1C3, pBac
[3xP3-EGFP]-m4B7 or pBac [3xP3-dsRed]-m2A10 plasmids with
the piggyBac helper plasmid was performed as described
previously, except that 0.1 mM p- nitrophenyl p′-guanidinobenzoate was
omitted from isotonic buffer [41]. Each G0
male was mated with 15 virgin females and groups of 5–10 G0
females were mated with 5 males, and G1 progeny were screened as
larvae with UV-fluorescence microscopy for the presence of the marker genes.
Standard Southern blotting and hybridization techniques were used to detect
transgene integration [42]. Genomic DNA was extracted from groups of six
transgenic or wild-type control females as described previously, except that DNA
pellets were re-suspended in 100 µl of dH2O [43]. The probe
used to identify m1C3 integration was amplified from a plasmid thought to
contain the EGFP ORF, but which in fact contained the ECFP ORF. These two ORFs
share 99% nucleotide sequence identity, so it is likely that the ability
of the probe to hybridize to the integrated gene was affected negligibly. The
EGFP probe was generated from pMos[3xP3-EGFP] [37] using
XbaI and SacI enzymes. The m4B7 probe was
generated through a restriction digest of the pBac [3xP3-EGFP]-m4B7
plasmid with both NaeI and FseI. The m2A10
probe was generated through a restriction digest of the pBSK-m2A10 plasmid with
both BamHI and BstBI. Probes were labeled with
32P using the Megaprime DNA labeling system (Amersham).

RT-PCR

Total RNA was isolated from whole or dissected mosquitoes using Trizol
(Invitrogen). For m2A10 RT-PCR analyses, 10 males or 2–3 whole females
were used for each RNA preparation. One microgram of RNA was treated with DNAseI
(Promega) for each 50 µl RT-PCR reaction. For m4B7 and m1C3 RT-PCR
analyses, 6 males, 4–15 female midguts, or 4 female carcasses were used
for each RNA preparation. Two hundred fifty nanograms of RNA were treated with
DNAseI for each 12.5 µl RT-PCR reaction. Gene-specific primers and a
OneStep RT-PCR Kit (Qiagen) were used for amplification of diagnostic products
from m2A10, m4B7, m1C3,
AsCPA[32],
AsVg1[23], or
An. stephensi ribosomal protein S26 gene [23]
transcripts (Table S2). For m2A10 RT-PCR analyses, amplification of diagnostic
products from AsVg1 and ribosomal protein S26 gene-specific primers was
performed in a single reaction. Diagnostic amplification reactions for the m4B7
25.1 and m1C3 P4.1 lines were initiated with one cycle at 50°C for 30 m, one
cycle at 95°C for 15 m, 32 cycles denaturation at 94°C for 30 s,
annealing at a reaction-specific temperature (Table S2)
for 30 s, and extension at 72°C for 1 m, followed by a final extension at
72°C for 10 m. Diagnostic amplification reactions for the m2A10 44.1 line
were performed as described, except that 30 cycles of amplification were
completed. For each sample, an additional control RT-PCR reaction tested for the
presence of genomic DNA contamination using ribosomal protein S26 gene primers
but omitting the reverse transcription step. Multiple biological replicates
(≥2) were performed for selected time points for each of the RT-PCR series of
experiments.

Real-time quantitative RT-PCR analysis

Female mosquito midguts and carcasses and male midguts were dissected in
phosphate-buffered saline (PBS), homogenized in Trizol Reagent (Invitrogen), and
total RNA extracted. Midguts were dissected at different time points (4 h, 8 h,
16 h, 24 h, 48 h, 72 h, 7 d, and 15 d) after a bloodmeal. RNA was treated with
DNase I (Invitrogen) at 1 U/µg RNA to remove potential genomic DNA
contamination. Further purification was performed using a DNA-free kit (Ambion).
A total of 0.4 µg of RNA was used for reverse transcription in a reaction
volume of 20 µl using ThermoScript RT-PCR System (Invitrogen). Real-time
quantitative PCR was performed on an Opticon 2 Real-Time PCR Detection System
using the Opticon Monitor software version 3.1 (Bio-Rad laboratories). m1C3
expression was quantified with Platinum SYBR Green qPCR SuperMix UDG with ROX
(Invitrogen) using gene-specific primers (Table S2)
to amplify a diagnostic fragment 211 bp in length. A series of quantitative
standards were generated from serial 10-fold dilutions (a range of
1010-1 molecules) of TOPO-m1C3scFv, in which full-length m1C3 was
cloned. Each assay was run in triplicate wells in a 25 µl final reaction
volume containing 2.5 µl of Platinum SYBR Green, 400 nM each forward and
reverse primer, and 2.5 µl cDNA sample. Each run included negative
controls consisting of wild-type control cDNA and water instead of cDNA. The
amplification protocol consisted of 2 min at 50°C, 2 min at 95°C,
followed by 40 cycles of amplification (94°C for 15 s, 60°C for 45 s,
plate read of SYBR Green I fluorescence), after which a melting-curve reaction
was conducted from 42°C to 95°C with plate readings every 1°C.
GraphPad Prism software was used to calculate statistical significance using
paired T-tests.

Immunoblot analysis

Mosquitoes were blood-fed on chickens and homogenized in a protease inhibitor
solution made from complete mini (Roche) and Pefabloc SC (Roche). An equal
volume of Laemmli sample buffer (Bio-Rad) with 0.1 M dithiothreitol was added.
Homogenates were separated on a 12% Tris-HCl polyacrylamide gel in
1×Tris/Glycine/SDS buffer (Bio-Rad), transferred to Immun-Blot PVDF
membrane (Bio-Rad), and incubated with goat anti-E tag polyclonal antibody
conjugated to horse radish peroxidase (Abcam). ECL Plus Western Blotting
Detection Reagents (GE Healthcare) were used to detect bound antibody. Ten
females were used for each hemolymph sample preparation. Legs were removed with
forceps and the proboscis was cut with a scissor. Individuals were inserted into
a pipette tip plugged with glass wool and threaded through a 0.5 ml tube placed
in a 1.5 ml collection tube. Centrifugation at 530 g for 10 min at 4°C
extracted hemolymph. Each hemolymph sample was mixed with 25 µl 0.15 M
NaCl and centrifuged at 2040 g for 5 min at 4°C. Fifteen microliters of the
middle fraction of the sample was transferred to a new 1.5 ml tube, to which 10
µl of the protease inhibitor solution was added. For immunoblots with
non-denatured samples, native sample buffer (Bio-Rad), 4–15%
Tris-HCl polyacrylamide gels (Bio-Rad), 1×Tris/Glycine electrophoresis
buffer (Bio-Rad), and native transfer buffer (25 mM Tris, 25 mM Glycine, pH 9.2)
were used.

Parasite challenge experiments

Four to six day-old transgenic and wild type female mosquitoes were fed with
P. falciparum NF 54 gametocytes using a membrane feeding
apparatus. After 15 min of feeding, un-engorged mosquitoes were removed and
engorged mosquitoes were maintained in the insectary under standard conditions
[37]
with daily access to a 10% sucrose solution or water and raisins. Midguts
were dissected 9 days after the infectious bloodmeal, stained with 0.1%
mercurochrome and the number of oocysts in each preparation counted. Uninfected
bloodmeals were provided to transgenic and wild-type control mosquitoes
following the membrane feeding. For the m4B7 25.1 experiments, mosquitoes were
allowed to feed on the first and second days post-infection. Mosquitoes in the
m2A10 experiments were allowed to feed on the 4th, 8th,
and 12th days post-infection. Engorged and un-engorged females were
not separated after the uninfected bloodmeals in m2A10 experiments 1–4,
while un-engorged females were discarded in experiments 5–7. Samples of
wild-type control and m2A10 females were dissected for oocyst counts on the
10th day post-infection. The salivary glands of all remaining
m2A10 and wild-type control females were dissected 17–19 days
post-infection. A hemacytometer was used in m2A10 experiments 1–4 to count
salivary gland sporozoites [13]. The samples in
experiments 5–7 were dried on 6 mm well slides and stored at
−20°C. Sporozoites were stained using SlowFade Gold antifade reagent
with DAPI (Invitrogen) and counted with a Zeiss Axioskop using the Axiovision
camera and software. Sporozoites were counted using one of three methods,
depending on parasite density. Method 1: If the number of sporozoites in each of
five fields was counted, and a total of 3 or more sporozoites was found, an
average sporozoite/mm2 measurement was calculated. When a field
contained greater than 50 parasites, Improvision Volocity software was used to
count the number of sporozoite nuclei in the DAPI image (Measurement protocol:
1. Find 2D nuclei: separate touching nuclei with a separation guide of 0.4
µm, reject nuclei with an area of less than 0.2 µm2. 2.
Exclude objects by size: exclude objects >10 µm2). Method 2:
If the 3 sporozoite requirement of method 1 was not met, fields were examined
until 3 sporozoites were counted, and an average sporozoite/mm2
measurement was calculated. Sporozoite/mm2 values were used to
calculate the total number of sporozoites present in the 6 mm2 slide
well area. Method 3: If a total of 3 sporozoites was not found in up to 25
fields, the entire 6 mm2 slide well area was examined for an exact
count. GraphPad Prism software was used to calculate statistical significance
using Mann-Whitney U tests.